Fig. 6

Requirement of scleraxis for Smad3-mediated gene expression. a Over-expression of Scx (AdScx) for 24 h rescues expression of vimentin, αSMA, ED-A fibronectin and fibrillar collagen mRNA (by qPCR) in primary cardiac proto-myofibroblasts obtained from Scx null mice compared to controls (AdGFP); n = 3 independent samples per genotype. Impaired collagen expression in Scx null cells is thus due to Scx loss and not a defect in the basal transcriptional machinery. b Up-regulation of fibrillar collagen mRNA expression by Smad3 (assessed by qPCR) is attenuated following Scx knockdown (AdshScx) compared to shRNA control in cardiac proto-myofibroblasts (AdshLacZ); n = 3. c Scx and Smad3 physically interact. Expression vectors for HA-tagged Scx and myc-tagged Smad3 were individually or jointly transfected into NIH-3T3 fibroblasts, then immunoprecipitated (IP) by anti-HA antibodies and subjected to western blotting with anti-myc antibodies (upper panels). Non-immunoprecipitated input and non-specific IgG antibodies were employed as positive and negative controls, respectively. Equal loading of transfected whole-cell lysates is shown by anti-HA and anti-myc western blots. Conversely, co-immunoprecipitation with Smad3 was not observed when a Scx mutant lacking its protein-interaction domain (HA-ScleraxisΔHLH) was employed (lower panels). d Smad3 and RNA polymerase II interaction with the Col1α2 gene promoter is significantly impaired in cardiac fibroblasts derived from Scx KO mouse hearts compared to WT, as assessed by chromatin immunoprecipitation (anti-Smad3 antibody vs non-specific IgG antibody) and re-ChIP (anti-RNA Pol II antibody vs non-specific IgG antibody) followed by qPCR, indicating that Scx loss impairs Smad3 signaling and Smad3-mediated recruitment of the transcription complex to the Col1α2 gene promoter; n = 3 independent samples per genotype. e Adenovirus-mediated over-expression of a DNA-binding Scx mutant (AdScxΔBD; 10, 50 or 100 MOI) in human ventricular myofibroblasts attenuates Smad3 and RNA polymerase II recruitment to the Smad-binding element of the Col1α2 gene promoter compared to control (AdGFP) performed as in (d); n = 3. *P < 0.05 vs WT (a) or vs AdGFP + AdshLacZ (b) or vs IgG (d–e), #P < 0.05 vs KO + AdGFP (a) or vs AdSmad3 + AdshLacZ (b) or vs WT (d) or as indicated (e)